Dopamine (DA) is a key transmitter in motor, cognitive, and reward pathways of the brain, with dysfunction of DA transmission linked to significant disorders, including Parkinson's disease, schizophrenia, and addiction. The long-term goal of this project is to identify local factors that regulate somatodendritic DA release from DA neurons in the substantial nigra pars compacta (SNc) and ventral tegmental area (VTA) and axonal DA release in striatum. Our previous work focused on release regulation by endogenous glutamate, GABA, and Ca2+ entry, using real-time voltammetric recording of evoked DA release. We discovered that hydrogen peroxide (H202), a reactive oxygen species (ROS) is in intracellular messenger in SNc DA neurons, that both modulates cell firing rate and inhibits somatodendritic DA release. In dorsal striatum, H202 is a diffusible messenger that mediates regulation of axonal DA release by glutamate and GABA. These effects are mediated by H2O2-dependent activation of ATP-sensitive K+ (K-ATP) channels. Proposed work will provide mechanistic insight into regulation of DA transmission by H202, as well as indicate functional consequences of H2O2 signaling on somatodendritic and axonal DA release. Aim 1 will test the hypothesis that H2O2 activates K-ATP channels by decreasing channel sensitivity to ATP; Aim 2 will determine the ionic dependence of H2O2 generation; Aim 3 will investigate the role of H2O2 generation in reguaiton of somatodendritic DA release and DA cell physiology by glutamatergic NMDA receptors; and Aim 4 will evaluate the temporal and spatial characteristics of glutamate-dependent H202 signaling in dorsal striatum. Methods include voltammetric detection of DA release, whole-cell and excised patch recording, and fluorescence imaging of H2O2, intracellular ions, and mitochondria! potential, using transfected cells, isolated neurons, and brain slices from guinea pigs and K-ATP channel-subunit knockout mice. Several brain disorders that involve dopamine dysfunction, including Parkinson's disease and schizophrenia, have also been linked to oxidative stress. Proposed studies will clarify how endogenous hydrogen peroxide normally regulates dopamine release. Because unregulated H2O2 can lead to oxidative stress, however, the findings may also point to possible targets for therapeutic intervention in these disorders.